A novel chitinase from the earthworm,Eisenia andrei

A novel chitinase gene, EaChi, and its expression pattern from the earthworm, E. andrei are demonstrated. Based on a deduced amino acid sequence, in EaChi, two specific domains for GH family 18 are well conserved with two essential amino acid residues for enzyme activity. The phylogenetic analysis shows that earthworm chitinase, EaChi, is evolutionarily close to other lophotrochozoan chitinases. The expression pattern analysis of EaChi indicates that the major expression is localized at intestinal epithelium and epidermis, possibly suggesting that the prime functions of the chitinase activity could be related to not only digestive process but also self-defending immunity as a biochemical barrier to protect the invasion of chitin-containing pathogens, including fungi, nematodes and protozoa.     

Non-inducibility of the glutathione transferases of the earthworm Eisenia andrei

1. Earthworms were exposed to solutions of potential biotransformation enzyme inducers, i.e. trans-stilbene oxide (0.1 mM), 3-methylcholanthrene (50 μM) or 1,4-bis[2-(3,5-dichloropyridoxyl)]benzene (30 μM) for 1 hr each day in three successive days.2. Glutathione transferase (GST) was determined in cytosolic extracts of the worms the day after the last treatment, with chloro-2,4-dinitrobenzene, l,2-dichloro-4-nitrobenzene and ethacrynic acid as electrophilic substrates. Activity toward 1,2-epoxy-3-[p-nitro-fenoxy]propane was not detected.3. With all three substrates, the specific activity in the cytosol was similar in treated animals and controls.4. Ion exchange chromatography was performed on a NeoBar AQ^TMcolumn (The “Low Pressure High Performance” technique), and GST eluted in four distinct peaks.5. The elution profiles of GST activity were almost identical for exposed earthworms and controls.6. The results suggest that earthworms may not have inducible GST, perhaps because they are detritus eaters, and do not need to detoxicate poisonous secondary plant metabolites.     

In vitro characterization of cholinesterases in the earthworm Eisenia andrei

Assessment of pollution impact in soil ecosystems has become a priority and interest has grown concerning the use of invertebrates as sentinel organisms. Inhibition of cholinesterase (ChE) activity has a great potential as a biomarker of pesticide exposure, and we evaluated the ChE kinetic parameters in the earthworm Eisenia andrei in the presence of acetylthiocholine (ASCh), proprionylthiocholine (PSCh) and butyrylthiocholine (BSCh). The highest ChE activity was found in the presence of ASCh and PSCh (42.45 and 49.82 nmol min(-1) mg protein(-1), respectively). BSCh was hydrolyzed at a rate of 4.04 nmol min(-1) mg protein(-1), but the time course did not reach a plateau under our experimental conditions. Km values were 0.142+/-0.006 and 0.183+/-0.053 mM for ASCh and PSCh, respectively. ASCh and PSCh hydrolysis were significantly inhibited by eserine (IC50 values were 1.44 x 10(-8) and 1.20 x 10(-8) M, respectively) and by carbaryl (IC50 values of 5.75 x 10(-9) and 4.79 x 10(-9) M). The presence of different ChEs in tissues from E. andrei was assessed by using selective inhibitors for AChE (BW284c51) and BChE (iso-OMPA). BW284c51 strongly reduced ASCh and PSCh hydrolysis and slightly affected that of BSCh, while iso-OMPA was without effect in all cases.     

Transcriptome analysis in the midgut of the earthworm (Eisenia andrei) using expressed sequence tags

In order to gain insight into the expression profiles of the earthworm midgut, we analyzed 1106 expressed sequence tags (ESTs) derived from the earthworm midgut cDNA library. Among the 1106 ESTs analyzed, 557 (50.4%) ESTs showed significant similarity to known genes and represented 229 unique genes of which 166 ESTs were singletons and 63 ESTs manifest as two or more ESTs. While 552 ESTs (49.9%) were sequenced only once, 230 ESTs (20.8%) appeared two to five times and 324 ESTs (29.3%) were sequenced more than five times. Considering this redundancy of expression, it is likely that the gene expression profile of the earthworm midgut would be polarized. The expression of globin-related proteins, including ferritin and linker chain, and fibrinolytic enzymes appeared to account for 10.1% and 4.7% of the total ESTs analyzed in this study, respectively. This suggests that the prime functions of the midgut in the earthworm would be associated with protein hydrolysis as well as globin formation. Among the recognized protein-coding genes, the gene category involved in protein synthesis appeared to be the largest one accounting for 15.6% of the expression in the midgut, followed by gene categories associated with energy (11.2%), homeostasis (10.8%), metabolism (3.6%), cytoskeleton (2.5%), and protein fate (1.4%). With regard to functional aspects, the most abundantly expressed genes were associated with respiratory pigment (10.1%), cellular respiration (8.6%), and fibrin hydrolysis (4.7%). In addition, we were able to identify novel ESTs in the earthworm, which were related to the innate immune system, including destabilase, a possible antagonist of transglutaminase.     

Application of a biomarker battery for the evaluation of the sublethal effects of pollutants in the earthworm Eisenia andrei

We applied a battery of biomarkers in the earthworm Eisenia andrei to assess the pollutant-induced stress syndrome. Earthworms were exposed to sublethal concentrations of copper (1-10 microM) and benzo[a]pyrene (0.01-10 microM) as models of inorganic and organic pollutants for 1, 3 and 7 days. Four potential biomarkers of stress were measured: lysosomal membrane stability of coelomocytes, lysosomal accumulation of lipofuscin in chloragogenous tissue and of neutral lipids in coelomatic cells, plasma membrane Ca2+-ATPase activity in the intestinal epithelium; metallothionein content was also evaluated as a biomarker of exposure. Significant changes were observed in the parameters measured in earthworms exposed to both contaminants. Certain biomarkers, such as lysosomal membrane stability and Ca2+-ATPase activity, that showed a decreasing trend with increasing pollutant concentration and time of exposure, proved to be particularly suitable to describe the evolution of a stress syndrome from its early phase to the development of pathological conditions. On the other hand, contaminant-induced changes in lysosomal lipofuscin and neutral lipid accumulation showed a bell-shaped trend, indicating that these biomarkers are able to follow the development of pollutant-induced stress syndrome as far as an equilibrium in the cell functions is maintained; therefore, they are particularly useful to describe mild stress conditions.     

Acetylcholinesterase activity in the earthworm Eisenia andrei at different conditions of carbaryl exposure

Recent reports have stressed the need for a better understanding of earthworm biomarker responses. We aimed at investigating acethylcholinesterase (AChE) activity in the earthworm Eisenia andrei after exposure to carbaryl or its commercial formulation Zoril 5 under different in vitro and in vivo experiments. In addition, lysosome membrane stability was assessed by neutral red retention assay in the same experimental conditions. AChE basal Km and Vm values were about 0.16 mM and 41 nmol min(-1) mg protein(-1), respectively. Carbaryl dose-dependently decreased Vmax, while not affecting Km values. Carbaryl reduced earthworm AChE activity within 1 day of in vivo exposure to contaminated filter paper. Tested on soil, carbaryl inhibited AChE with the maximum effect after 3 days; in contrast, lysosome membrane stability of coelomocytes indicated a maximum toxicity after one day, followed by a recovery. AChE inhibition by Zoril 5 was highest after one day, while lysosome membrane stability declined progressively. In all cases, carbaryl dose-dependently decreased Vmax while not affecting Km values. In conclusion, E. andrei AChE activity assessed in vitro is dose-dependently inhibited by the carbamate compound carbaryl, which acts as a pure competitive inhibitor. In vivo experiments suggested that pure and co-formulated carbaryl have different time and/or dose dependent effects on earthworms. Our results further support the use of AChE inhibition as an indicator of pesticide contamination, to be included in a battery of biomarkers for monitoring soil toxicity.     

Evidence of lipofuscin and melanin in the brown body of the earthworm Eisenia fetida andrei

By means of histochemical and electron-microscopic techniques, two pigments were identified in the brown body of the earthworm Eisenia fetida andrei. Lipofuscin, often called the aging pigment, was the more abundant pigment and the first to be synthesized. Melanin was also found but was only present in the mature brown body. A computer-assisted image analysis was used to quantify the relative abundance of the two pigments at different stages of the formation of the brown body. A previous study showing phenoloxidase activity in the coelomocytes and the present demonstration of the ability of these cells to produce reactive oxygen systems when agglutinated can be correlated with the simultaneous presence of the two pigments in the brown body. The physiological significance of pigment synthesis by brown bodies is discussed with respect to the immune response in earthworms.     

Enzymatic resolution of amino acids via ester hydrolysis

Summary The present review outlines recent examples of enzyme-based resolution procedures for amino acids via the hydrolysis of their esters. The resolutions have been achieved by using proteases (-chymotrypsin, subtilisin and other microbial proteases, and sulfhydryl proteases of plant origin) and lipases. Relevant work utilizing yeast and other microbial cells is also included.     

Multiple mating increases cocoon hatching success in the earthworm Eisenia andrei (Oligochaeta: Lumbricidae)

In simultaneous hermaphrodites with reciprocal mating, multiple mating may be a male strategy that conflicts with female interests, and therefore an intra‐individual sexual conflict regarding the number of matings may be expected. The evolutionary outcome of this sexual conflict will depend on the costs and benefits that extra mating entails for each sexual function. In the present study, we investigated the costs and benefits of multiple mating on cocoon number, cocoon mass, and cocoon hatching success in the redworm Eisenia andrei, a simultaneous hermaphrodite with reciprocal insemination, by manipulating the number of matings with different partners. We did not detect any reduction in the female reproductive output (number and mass of cocoons) with increasing number of mating partners. However, we found that multiple mating showed benefits for female reproduction that increased the hatching success of the cocoons. This effect may be a result of increased quantity and/or diversity of sperm in the spermathecae of multiple mated earthworms. Further studies are required to clarify the mechanism underlying the increased cocoon hatching success when redworms engage in multiple matings. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ?? , ??–??.     

Enzymatic hydrolysis of cellulosic municipal wastewater treatment process residuals as feedstocks for the recovery of simple sugars

This study examined the hydrolysis of lignocellulose extracted from municipal wastewater treatment process residuals for the purpose of investigating low-cost feedstocks for ethanol production, while providing an alternative solid waste management strategy. Primary and thickened waste activated sludges and anaerobically digested biosolids underwent various pre-treatments to enhance subsequent enzymatic hydrolysis. Half of the pre-treated samples were dried and grinded, while the other half were used as is (wet). The wet primary sludge yielded the highest reducing sugar conversions. When wet primary sludge without pre-treatment was hydrolyzed at 40 °C and an enzyme loading of 800 U/g substrate, 31.1 ± 2.7% was converted to reducing sugars in 24 h. This increased to 54.2 ± 4.0% when HCl and KOH pre-treatments were applied. FTIR analyses were used to examine differences in the sludge compositions. These indicated that the cellulose content in the primary sludge was higher than that of the thickened waste activated sludge and biosolids, which was consistent with the higher reducing sugar yields observed in the primary sludge.     

Porcine pancreatic lipase-catalized enantioselective hydrolysis of N-protected amino acid methyl-esters

Summary A preparative-scale enantioselective hydrolysis of racemic methyl esters of several N-protected amino acid has been carried out by using crude porcine pancreatic lipase (Triacylglycerol lipase, EC 3.1.1.3) PPL as a hydrolytic enzyme. In all cases 50% of the racemic methyl ester was hydrolysed to the N-protected L-amino acid with high yield and high optical purity.Hydrolysis rates were very close related not only to the amino acid structure but also to the steric and/or electronic nature of the ester and N-protecting groups. Thus, the very convenient ester methyl group can be enantioselectively hydrolysed with PPL when N-protecting group is a carbonyl derivative, as it is the usual benzoyl group.     

Ontogenetic change in relative performance of allozyme genotypes influences detection of heterosis in the earthworm Eisenia andrei

The effect of ontogeny on relationships between allozyme genotypes and fresh weight was measured weekly throughout the life history of the earthworm Eisenia andrei to test the hypothesis that there is an ontogenetic component to variation in such relationships. Two of six allozyme loci showed a significant increase in apparent heterosis with ontogeny, while one locus showed a significant decrease in apparent heterosis. Three loci showed a significant decrease in the performance of common homozygotes with ontogeny. Patterns of relative genotypic performance varied among loci, but the cumulative effect was an increase in apparent allozyme heterosis later in ontogeny coinciding with a series of positive relationships between multilocus heterozygosity and fresh weight. The results could not be used to determine whether these patterns were caused by selection acting on the loci directly or on loci tightly linked to allozyme loci. However, because the same individuals were used throughout this study and thus allele frequencies and heterozygote deficiency were constant, the presence of both ontogenetic effects and differences in such patterns among loci is not compatible with a general inbreeding effect. Examining relative genotypic performance repetitively using the same individuals through ontogeny or in different environments is a very powerful experimental design for testing the effects of inbreeding or other populational factors.     

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and β-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external β-xylosidase.     

一种新型蚯蚓纤溶酶的部分性质研究

为获得一种高效的溶栓药物。从赤子爱胜蚓(Eiseniafoelida)中分离纯化得到了一种纤溶酶组分。用Lowry法测定蛋白质浓度,SDSPAGE鉴定纯度为98%,表观相对分子质量(Mr)为14850,纤维蛋白平板法测定其总纤溶活性为65.51×103mm2/mg,直接纤溶活性为15.61×103mm2/mg,间接纤溶活性为26.34×103mm2/mg。水解BAEE的米氏常数(Km)为1.82×105mol/L。水解ChromozymPL的米氏常数(Km)3.98×105mol/L,水解ChromozymtPA的米氏常数(Km)5.55×105mol/L活性,N端氨基酸序列测定的结果为VIGGTNAIPGEFPYQ。结果表明该纤溶酶分子量较小,间接活性较高,适宜作为一种新型的溶栓药物。     

Enzymatic hydrolysis of whey proteins: I. Kinetic models

We have studied the enzymatic hydrolysis of whey proteins at pH 8 and50 degrees C with two proteases of bacterial origin, MKC Protease 660 L, and one of animal origin, PEM 2500 S. Our results show that a greater degree of hydrolysis is achieved under the same experimental conditions with the bacterial proteases than with the animal one. In our interpretation of the results we propose a mechanism in which the hydrolytic reaction is a zero-order one for the substrate, and the enzyme denaturalizes simultaneously via a second-order kinetic process due to free enzyme attacking enzyme bound to the substrate. Our results also indicate that there is an irreversible serine-protease inhibitor in whey proteins. (c) 1994 John Wiley & Sons, Inc.     

Process development for the enzymatic hydrolysis of food protein: Effects of pre-treatment and post-treatments on degree of hydrolysis and other product characteristics

An enzymatic process was developed to produce protein hydrolysate from defatted soya protein. Various unit operations were tried, and the effects of pre- and post-treatments on the product characteristics such as degree of hydrolysis (DH), free amino acid content (%FAA) and average molecular weight (MW) were investigated. The use of acid washes showed no difference in %DH. Increasing pH during pre-cooking gave lower %DH. Alkaline cooking made too much insoluble protein, thus the protein yield was too small. A better hydrolysis with more acceptable taste was obtained when the combination of Neutrase/Alcalase/Flavourzyme was used in place of Alcalase/Flavourzyme combination. Untoasted defatted soya was more effective on the proteolysis than toasted one. The MW of the evaporated and spray dried product was higher than that of undried product, due to precipitation of low-solubility components. When the product separation was carried out by ultrafiltration and the product concentration by reverse osmosis, the solubility and the taste of the product were improved. The difference between enzyme hydrolysate and acid hydrolysate was significant in free amino acid composition, especially in tyrosine, phenylalanine, glutamine and asparagine.     

Enzymatic hydrolysis of plumieride to plumieridine

The hydrolysis of plumieride to form the aglycone plumieridine was catalysed by a commercial preparation of cellulase from Aspergillus niger.     

Gene expression profiling of coelomic cells and discovery of immune-related genes in the earthworm,Eisenia andrei,using expressed sequence tags

The coelomic cells of the earthworm consist of leukocytes, chlorogocytes, and coelomocytes, which play an important role in innate immunity reactions. To gain insight into the expression profiles of coelomic cells of the earthworm, Eisenia andrei, we analyzed 1151 expressed sequence tags (ESTs) derived from the cDNA library of the coelomic cells. Among the 1151 ESTs analyzed, 493 ESTs (42.8%) showed a significant similarity to known genes and represented 164 unique genes, of which 93 ESTs were singletons and 71 ESTs manifested as two or more ESTs. From the 164 unique genes sequenced, we found 24 immune-related and cell defense genes. Furthermore, real-time PCR analysis showed that levels of lysenin-related proteins mRNA in coelomic cells of E. andrei were upregulated after the injection of Bacillus subtilis bacteria. This EST data-set would provide a valuable resource for future researches of earthworm immune system.     

Characterization of two endoglucanases for the classification of the earthworm,Eisenia fetida Waki

Eisenia fetida and Eisenia andrei are vermicomposting species that are used as model animals for testing chemical material toxicology. Eisenia spp. are grown commercially in various fields in Japan. However, these two species have not been classified because it is difficult to distinguish them morphologically; thus, all bred earthworms are called E. fetida. However, it has been proposed that these two species have different expression regulation mechanisms. Here, we classified a sample of earthworms purchased from several farms, confirming that both E. fetida and E. andrei are present in Japanese earthworm breeding programs. We also characterized two highly active endoglucanases (EfEG1 and EfEG2) from the E. fetida Waki strain, which contained strong fibrinolytic enzymes for improving human health. We confirmed that EfEG1 is 1371 bp long and belongs to GHF9. Thus, E. fetida Waki may have commercial application for biomass utilization and as a dietary health supplement.